Evidence of oocyte polarity in bovine; implications for intracytoplasmic sperm injection and somatic cell nuclear transfer

Objective: We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine

Materials and Methods: In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to [HNS] and far-from [FS] spindle] or trisection [into MII-spindle [S], the spindle-side half [NS], and the distal half unassociated with the spindle [FS]]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction [RT-qPCR]. To map the possible preferential sperm entry point [SEP], the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization

Results: The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S [Tead4, Nanog, Ctnb and Sox2], NS [Oct4], or FS [Gata6]. The SEP in almost [90%] fertilized oocytes was located in MII-hemisphere

Conclusion: The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection [where a sperm is injected far from the MII-spindle] and somatic cell nuclear transfer [where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation]

Effect of ovarian cyclic status on in vitro embryo production in cattle

The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle [LF], 2. the presence of a corpus luteum [CL] and 3. ovaries without LF or CL [WLCF]. Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group [36.6%], but non-significantly higher than that of the ST group [45.7%]. The highest blastocyst rate belonged to the CL group [54.6%] which was significantly greater than the WLCF group [32.9%] and non-significantly higher than the LF group [52.4%]. There was no significant difference in blastocyst rates in the CL and LF groups. Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and noninvasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage

Study of human chondrocyte redifferntiation capacity in three-dimensional hydrogel culture

Articular cartilage tissue defects cannot be repaired by the proliferation of resident chondrocytes. Autologous chondrocyte transplantation [ACT] is a relatively new therapeutic approach to cover full thickness articular cartilage defects by in vitro grown chondrocytes from the joint of a patient. Therefore, we investigated the differentiation capability of human chondrocytes maintained in alginate culture. The cartilage specimens obtained from 50 patients who underwent total knee and hip operations at the teaching hospital of Isfahan University of Medical Sciences, Isfahan Iran. Isolated primary chondrocytes were first grown in monolayer cultures for 1 to 6 passages [each passage lasting about 3 days]. At each passage, monolayer cells seeded in alginate culture and investigated morphologically and immuno-cytologically for expression of cartilage-specific markers [collagen type II and cartilage-specific proteoglycans]. The chondrocytes from monolayer passages PI to P4 introduced in alginate cultures regained a chondrocyte phenotype. Cells were interconnected by typical gap junctions and after few days, they produced a cartilage-specific extracellular matrix [collagen type II and cartilage-specific proteoglycans]. In contrast, cells from monolayer passages P5 and P6 did not redifferentiate to chondrocytes in the alginate cultures. Chondrocyte culture was established for the first time in Iran. The alginate culture conditions promote the redifferentiation of dedifferentiated chondrocytes that have still a chondrogenic potential. This procedure opens up a promising approach to produce sufficient numbers of differentiated chondrocytes for ACT. Indeed, in some patients the harvested cells were used immediately and successfully for transplantation